Rapid Intact mass based multi-attribute method in support of mAb upstream process development.

N-linked glycosylation is a critical quality attribute for monoclonal antibodies (mAbs) as it affects product stability, immunogenicity, receptor binding and antibody effector function, clearance and half-life. It has been widely accepted that the glycosylation process is greatly impacted by several factors during bioreactor operations. Therefore, the timely acquisition of N-linked glycosylation information during cell culture process development is critical for the success of the endeavor. In this paper we describe a simple, rapid, and robust Multi-Attribute Method (MAM) based on intact mass analysis. This method, developed for an open access instrument, has been optimized for the analysis of light and heavy chains generated from dithiothreitol (DTT) reduction of intact mAbs sampled directly out of bioreactors. The N-linked glycosylation profile, identity confirmation of light chain and heavy chain, light chain glycation and non-glycosylated heavy chain (NGHC) can all be monitored by this method. Our results confirm that the N-linked glycosylation profile determined using Intact mass based MAM is comparable with a release glycan assay and LC-MS/MS peptide based MAM assay for the most abundant glycoforms. Furthermore, the results for the NGHC attribute determined with our method are comparable to results from capillary gel electrophoresis (CGE) and LC-MS/MS peptide based MAM.